TGF-beta1 is an inducer of apoptosis in hepatocytes. To explore the signaling pathway leading to TGF-beta1-induced apoptosis, the regulatory mechanisms controlling cell cycle progression were investigated using a TGF-beta1-sensitive hepatoma cell line, FaO. Cell cycle analysis demonstrated that the accumulation of apoptotic cells was preceded by a progressive decrease of the cell population in the G1 phase concomitant with a slight increase of the cell population in the G2/M phase in response to TGF-beta1. TGF-beta1 induced the transient increase in the mRNA and protein expression of Cdc2, cyclin A, cyclin B, and cyclin D1 at an early phase of apoptosis. During TGF-beta1-induced apoptosis, the transient increase in Cdks' kinase activities coincides with a dramatic increase in the hyperphosphorylated forms of RB. Both Cdc2 and Cdk2 kinase activated in response to TGF-beta1 bind to and phosphorylate RB. The treatment of roscovitine or olomoucine completely blocked TGF-beta1-induced apoptosis inhibiting Cdc2 and Cdk2 kinase from phosphorylating RB. These results suggest that Cdc2 plays a more critical role in TGF-beta1-induced apoptosis. In conclusion, we showed the first evidence that Cdc2 and Cdk2 kinase activity transiently induced by TGF-beta1 phosphorylates RB as a physiological target in FaO cells. The functional inactivation of RB following hyperphosphorylation may trigger abrupt cell cycle progression, leading to irreversible cell death. We also investigated the role of each mitogen-activated protein kinases (MAPK) in TGF-beta1-induced apoptosis. We demonstrate that strong sustained pro-apoptotic signals provided by both p38 and stress-activated protein kinase (JNK) are not overridden by counteracting extracellular signal-regulated kinase (ERK) activation during TGF-beta1-induced apoptosis. Also we demonstrated that p38 MAP kinase in TGF-beta1-induced apoptotic signaling pathways localizes downstream of Cdc2 and Cdk2, the critical signaling molecules for TGF-beta1-induced apoptosis. Furthermore, we provide the first evidence of the cross-talk between MAP kinases during TGF-beta1-induced apoptosis, suggesting that the integration and balance among MAP kinases may contribute to the final determination of cellular fate.